Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1225-5009(Print)
ISSN : 2287-772X(Online)
Flower Research Journal Vol.26 No.2 pp.61-67
DOI : https://doi.org/10.11623/frj.2018.26.2.04

Sucrose Concentration, Light Intensity, and CO2 Concentration Affect Growth and Development of Micropropagated Carnation

Ji Eun Park1, Luc The Thi1, Liu Ya1, Byoung Ryong Jeong1,2,3*
1Division of Applied Life Science (BK21 Plus Program), Gyeongsang National University, Jinju 660-701, Korea
2Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Korea
3Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, Korea
Corresponding author: Byoung Ryong Jeong Tel: +82-55-772-1913 E-mail: brjeong@gmail.com
20/03/2018 28/05/2018 17/06/2018

Abstract


Carnation (Dianthus caryophyllus L.) ‘Purple Beauty’ at micropropagation stage 3 was cultured under two levels each of medium sucrose concentration (0 and 30 g·L-1), photosynthetic photon flux density (50 and 200 μmol·m-2·s-1 PPFD), and CO2 concentration (350 and 1,000 μmol·mol-1), in a factorial experimental design, consisting of all eight possible treatment combinations. Shoot tip explants, obtained from in vitro-grown plantlets, were cultured on 50 mL agar-solidified Murashige and Skoog medium per container. Each culture container was sealed with a rigid lid vented by a high-efficiency particulate air filter attached to a 10 mm diameter hole made in the lid center, with an estimated number of 2.8 air exchanges per hour. All cultures were maintained for four weeks at 24°C/22°C (day/night) temperatures, 70%–80% relative humidity, and a 16 h/8 h (day/night) photoperiod was provided by white light-emitting diodes. The treatment combining high light intensity (200 μmol·m-2·s-1), high CO2 concentration (1,000 μmol·mol-1), and without supplementation of sucrose to the medium (i.e., photo-autotrophic conditions) resulted in better plantlet growth and development, with higher values being observed in terms of total fresh weight, tissue water content, leaf length, leaf width, and total chlorophyll content than in plantlets grown under the other treatments.



초록


    Rural Development Administration
    PJ01090805

    Introduction

    Carnation is one of the most important species in the worldwide market of cut flowers (Birlanga et al. 2015). Recently domestic production and cultivation area of carnation has been decreasing in South Korea, while the amount of import and the royalty paid to foreign carnation cultivars have continued to increase from the past decade (Ministry of Agriculture, Food and Rural Affairs Database 2015). In an effort to solve these problems, the Rural Development Administration has bred many domestic cultivars. Although growers are not quite satisfied with the performance of the new domestic cultivars in terms of flower quality and productivity, there have not been studies to obtain high quality propagated plants of these cultivars. Carnation is mainly propagated by cutting and production of propagated plants by cutting method in Korea is estimated to occupy about 20% of the cultivated area (Lee et al. 2006). However, it is desirable to use micropropagated virus-free plants, especially for those to be used as mother plants.

    Tissue culture or micropropagation is powerful technique for rapid propagation of physiologically uniform and healthy plantlets which can’t be done by seed propagation or other vegetative propagation method of low efficiency in limited period of time (Jeong et al. 1995; Murch et al. 2000; Pretto and Santarem 2000; Zobayed and Saxena 2004). However, the widespread use of micropropagation is limited in some situations due to high production costs. These costs are mostly attributed to a low growth rate, a significant loss of plantlets during in vitro tissue culture process due to microbial contamination, physiological and morphological disorders, poor rooting, low survival rate at the ex vitro acclimatization stage, and high labor costs (Afreen-Zobayed et al. 2000; Jeong et al. 1996; Kozai 1991; Xiao et al. 2011). In order to overcome these problems, the photo-autotrophic system has shown to be worthy to consider a suitable technique due to its advantages (Kozai 1991).

    Plantlets grown in the photo-autotrophic system can synthesize carbohydrates by utilizing CO2 in the atmosphere, defused into the tissue culture vessel through air membrane, so that the system enables the plantlets to be ready for the acclimatization step ex vitro. The photo-autotrophic system has many advantages in terms of fast growth rate of plantlets, few physiological disorders, high survival rate of plantlets, and low cost even though there are differences among plant species (Hwang and Jeong 2012; Ticha et al. 1998). The growth rates of plantlets of potato (Kim et al. 1999; Kozai et al. 1988a), strawberry (Fujiwara et al. 1988; Kozai and Sekimoto 1988; Kozai et al. 1988b), tobacco (Kozai et al. 1990b; Mousseau 1986), Phalaenopsis (Doi et al. 1989), and gerbera (Jeong et al. 1999) were cultured on the medium without supplementation of sucrose under high level of CO2 and/or high light intensities were greater than those were cultured on the medium with supplementation of sucrose under low levels of CO2 and low light intensities. Maintaining properly the in vitro environmental condition is critical to promote plantlet growth by increasing metabolic processes. In this study the effect of condition with different levels of medium sucrose, light intensity, and air CO2 has been investigated in carnation which are very prone to hyperhydricity to see how plantlet growth under photo-mixotrophic and photo-autotrophic culture conditions are affected.

    Materials and Methods

    Plant materials and culture conditions

    Shoot explants of carnation (Dianthus caryophyllus L.) obtained from in vitro-grown plantlets were stuck into and cultured on the MS medium (Murashige and Skoog 1962). The pH of all media was adjusted to 5.70 prior to autoclaving at 121℃ for 20 min. Media in the quantity of 50 mL per container were supplemented with one of the two levels of sucrose, 0 and 30 g·L-1, and were solidified with 0.8% agar (w/v) in 85 mm (diameter) x 110 mm (height) round plastic containers (Gaooze 0811C, Korea Scientific Technique Industry Co., Ltd., Suwon, Korea).

    Each container was capped with a lid which had a 10 mm diameter round hole made and sealed by a sheet of HEPA filter (Nihon Millipore Ltd., Yonezawa, Japan), with an estimated number of air exchanges per hour of 2.8. All the cultures were maintained in culture rooms at 24℃/22℃ (day/night) temperatures and 70-80% relative humidity (RH). Cultures were subjected to a light intensity of 50 or 200 μmol·m-2·s-1 photosynthetic photon flux density (PPFD), measured at the top of culture containers, for a 16 h photoperiod provided by white light emitting diodes (LED, 400-700 nm, PSLED-1203-50A, Force Lighting Co., Ltd., Hwaseong, Korea), and CO2 concentration of 350 or 1,000 μmol·mol-1 in culture rooms (Fig. 1). The experiment was set up in a completely randomized design and each treatment consisted of 3 replicates of 4 explants in each of two containers. All the measurements were taken in triplicates, totally 24 shoot explants per treatment, to verify the reproducibility of the results.

    Measurements of growth and morphological parameters

    After four weeks of culture, shoot length, number of nodes, root length, number of roots, fresh and dry weights of shoot, root and whole plantlet, tissue water content, leaf length, and leaf width were measured. Shoot, root, and total dry weights were measured a fter 72 h of d rying in a 70°C oven (FO-450M, Jeio Technology Co., Ltd., Daejeon, Korea).

    Measurements of total chlorophyll content

    For total chlorophyll content measurement, fresh leaf tissues (the same 20 mg) taken from the third leaf from the top in each treatment were extracted with 80% (v/v) acetone for 24 h. Total chlorophyll content was determined by measuring the absorbance at 645 and 663 nm by a spectrophotometer (Uvikon 992, Kotron Instrumentals, Milano, Italy) according to the method of Arnon (1949).

    Statistical analysis

    Data collected were analyzed for statistical significance by the SAS (Statistical Analysis System, V. 9.1, Cary, NC, USA) program. The experimental results were subjected to an analysis of variance (ANOVA) and Duncan’s multiple range tests at a p ≤ 0.05 probability level. Graphs were plotted with the SigmaPlot 10.0 (Systat Software Inc., San Jose, CA, USA).

    Results and Discussion

    The supplementation of sucrose to the medium significantly influenced the shoot length, number of nodes, root length, shoot and total fresh weights, dry weights of shoot, root and whole plantlet, and tissue water content (Table 1). Light intensity significantly affected shoot length, number of nodes, number of roots, root and total fresh weights, shoot and total dry weights, tissue water content, leaf length, leaf width, and total chlorophyll content. The CO2 supplemented to the culture room also affected shoot length, number of nodes, total chlorophyll content, root length, shoot and total fresh weights, shoot and total dry weights, leaf length, and leaf width.

    Figure 2A shows the effect of level of medium sucrose, light intensity, and air CO2 on shoot length of carnation plantlets cultured in vitro for four weeks. Shoot length increased in the treatment without supplementation of sucrose as compared to those with supplementation of sucrose. Generally, a photo-autotrophic condition with CO2 supplementation to the culture room and high light intensity promoted the growth and development of the shoot, while it had little influence on the shoot length, thus resulting in more compact shoot growth. Similar results were obtained with number of nodes as the shoot length. The treatments without supplementation of sucrose to the medium showed decrease in the number of nodes when the cultures were supplied with higher light intensity along with a higher concentration of CO2. In contrast, in the photo-mixotrophic condition, number of node increased by a higher level of CO2 when the light intensity was increased (Fig. 2B).

    Root length also showed a similar trend as the shoot length. The treatment without supplementation of sucrose showed root length to increase, especially in the treatment of a low light intensity. Root length increased in the treatment of a lower light intensity in the photo-autotrophic condition. On the other hand, in the photo-mixotrophic condition, root length increased with increased light intensity (Fig. 3A). Deng and Donnelly (1993) obtained results that the photo-autotrophic condition producing plantlets with longer length of the longest root as compared to the photo-mixotrophic condition. The number of roots increased with increasing light intensity. The photo-autotrophic condition with a higher light intensity showed unexpected decrease in the number of roots with the increased CO2 concentration, which implied that 1,000 μmol·mol-1 CO2 was excessively too high for the young cultures (Fig. 3B).

    As for the fresh weight of whole plantlet, the photo-autotrophic condition gave better results than the photo-mixotrophic condition. Kozai et al. (1988a) also reported that the total fresh weight of carnation plantlets was greater under the photo-autotrophic than photo-mixotrophic condition. Total fresh weight increased with an increase in CO2 concentration and light intensity (Fig. 4A). Similar results were reported by Deng and Donnelly (1993), Nguyen and Kozai (2001), Xiao et al. (2007), and Zhang et al. (2009) who described that high light intensity and/or high CO2 concentration increased total fresh weight. Total dry weight showed very similar trend as the total fresh weight. However, the photo-mixotrophic condition gave better results than the photo-autotrophic condition (Fig. 4B). Similar results have been obtained for tobacco (Tichá et al. 1998). The tissue water content of the plantlets grown in the photo-autotrophic condition was significantly higher than that of the plantlets grown in the photo-mixotrophic condition. The tissue water content was slightly lower in plantlets cultured under a high than a low light intensity, indicating less succulent growth in this condition (Fig. 5A). Total chlorophyll content was significantly higher in the photo-autotrophic than in the photo-mixotrophic condition. Increasing CO2 concentration and light intensity increased the total chlorophyll content (Fig. 5B). Studies on increasing the total chlorophyll content of plantlets by increasing the concentration of CO2 in the culture room have been reported in tobacco (Mosseau 1986), cymbidium (Kozai et al. 1990a), potatoes (Cournac et al. 1992), and Limonium spp. ‘Ocean Blue’ (Jeong and Jeong 2002).

    Leaf l ength and leaf w idth s howed v ery similar trend each other. In the photo-autotrophic condition, leaf length and width were significantly increased. However, in the photo-mixotrophic condition, they significantly increased only under the high light intensity and high CO2 concentration (Fig. 6A and B).

    Overall, supplemented sucrose to the medium negatively affected the growth and development of carnation plantlets cultured in vitro. However, light intensity and CO2 concentration positively affected total fresh and dry weights, and total chlorophyll content, while slightly suppressing the shoot length. In conclusions, better growth and development of the plantlets were observed under the photo-autotrophic than photo-mixotrophic condition. The finding is in agreement with previous reports that the photo-autotrophic condition promotes the growth of various plant species as compared to the photo-mixotrophic condition (Kozai et al. 1991; Oh et al. 2009; Xiao and Kozai 2004; Zobayed et al. 1999).

    Acknowledgements

    This s tudy was c arried o ut w ith a support from the Korea Rural Development Administration (Project No. PJ01090805). Ji Eun Park, Luc The Thi, and Liu Ya were supported b y a s cholarship f rom the BK21 Plus P rogram, Ministry of Education, Republic of Korea.

    Figure

    FRJ-26-61_F1.gif

    The experimental set-up with two levels each of medium sucrose, light intensity, and air CO2.

    FRJ-26-61_F2.gif

    Effect of level of medium sucrose (g·L-1), light intensity (μmol·m2·s-1 PPFD), and air CO2 (μmol·mol-1) on shoot length (A) and number of node (B) of carnation plantlets cultured in vitro for four weeks. Vertical bars indicate ± standard errors. Lower case letters above each bar indicate statistically significant means according to the Duncan’s multiple range test (p ≤ 0.05).

    FRJ-26-61_F3.gif

    Effect of level of medium sucrose (g·L-1), light intensity (μmol·m-2·s-1 PPFD), and air CO2 (μmol·mol-1) on root length (A) and number of roots (B) of carnation plantlets cultured in vitro for four weeks. Vertical bars indicate ± standard errors. Lower case letters above each bar indicate statistically significant means according to the Duncan’s multiple range test (p ≤ 0.05).

    FRJ-26-61_F4.gif

    Effect of level of medium sucrose (g·L-1), light intensity (μmol·m-2·s-1 PPFD), and air CO2 (μmol·mol-1) on total fresh weight (A) and total dry weight (B) of carnation plantlets cultured in vitro for four weeks. Vertical bars indicate ± standard errors. Lower case letters above each bar indicate statistically significant means according to the Duncan’s multiple range test (p ≤ 0.05).

    FRJ-26-61_F5.gif

    Effect of level of medium sucrose (g·L-1), light intensity (μmol·m-2·s-1 PPFD), and air CO2 (μmol·mol-1) on tissue water content (A) and total chlorophyll content (B) of carnation plantlets cultured in vitro for four weeks. Vertical bars indicate ± standard errors. Lower case letters above each bar indicate statistically significant means according to the Duncan’s multiple range test (p ≤ 0.05).

    FRJ-26-61_F6.gif

    Effect of level of medium sucrose (g·L-1), light intensity (μmol·m-2·s-1 PPF D), and a ir CO2 (μmol·mol-1) on leaf length (A) and leaf width (B) of carnation plantlets cultured in vitro for four weeks. Vertical bars indicate ± standard errors. Lower case letters above each bar indicate statistically significant means according to the Duncan’s multiple range test (p ≤ 0.05).

    Table

    Results of significance test (F test) on growth and developmental parameters in carnation ‘Purple Beauty’ cultured in vitro.

    Reference

    1. F. Afreen-Zobayed , S.M.A. Zobayed , C. Kubota , T. Kozai , O. Hasegawa (2000) A combination of vermiculite and paper pulp supporting material for the photoautotrophic micropropagation of sweet potato., Plant Sci., Vol.157 ; pp.225-231
    2. D.I. Arnon (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris., Plant Physiol., Vol.24 ; pp.1-15
    3. V. Birlanga , J. Villanova , A. Cano , E.A. Cano , M. Acosta , J.M. PA(c)rez-PA(c)rez (2015) Quantitative analysis of adventitious root growth phenotypes in carnation stem cuttings., PLoS One, Vol.10 ; pp.e0133123
    4. L. Cournac , I. Cirier , P. Chagvardieff (1992) Improvement of photoautotrophic Solanum tuberosum plantlet culture bylight and CO2: Differential development of photosynthetic characteristics and varietal constraints., Acta Hortic., ; pp.53-58
    5. R. Deng , D.J. Donnelly (1993) In vitro hardening of red raspberry through CO2 enrichment and relative humidity reduction on sugar-free medium., Can. J. Plant Sci., Vol.73 ; pp.1105-1113
    6. M. Doi , H. Oda , T. Asahira (1989) In vitro atmosphere of cultured C3 and CAM plants in relation to day-lengths., Envrion. Control Biol., Vol.27 ; pp.9-13
    7. K. Fujiwara , T. Kozai , I. Watanabe (1988) Development of a photoautotrophic tissue culture system for shoots and/orplantlets at rooting and acclimatization stages., Acta Hortic., ; pp.153-158
    8. S.J. Hwang , B.R. Jeong (2012) Growth of strawberry plantlets cultured in vitro in the agar or commercial plug mediumas affected by ionic strength., Weonye Gwahag Gisulji, Vol.30 ; pp.201-207
    9. B.R. Jeong , K. Fujiwara , T. Kozai (1995) Environmental control and photoautotrophic micropropagation., Hortic. Rev. (Am. Soc. Hortic. Sci.), Vol.17 ; pp.125-172
    10. B.R. Jeong , C.S. Yang , E.J. Lee (1996) Photoautotrophic growth of Dianthus caryophyllus in vitro as affected by photosynthetic photon flux and CO2 concentration., Acta Hortic., ; pp.611-615
    11. G.W. Jeong , B.R. Jeong (2002) Autotrophic growth of Limonium spp. ?~Ocean Blue ?(tm) plantlets in vitro as affected by PPF, NAEH and CO2 concentration., J Bio-Environ Con, Vol.11 ; pp.115-120
    12. J.D. Jeong , H.S. Lee , C.G. Kim , Y.Y. Cao , K.B. Lim (1999) Effects of substrate, sucrose and CO2 concentration on in vitro multiplication and growth of Gerbera hybrida ?~Beauty ?(tm)., J Korean Soc Hort Sci, Vol.40 ; pp.477-480
    13. H.S. Kim , E.M. Lee , M.A. Lee , I.S. Woo , C.S. Moon , Y.B. Lee , S.Y. Kim (1999) Production of high quality potato plantlets by autotrophic culture for aeroponics systems., J Korean Soc Hort Sci, Vol.40 ; pp.26-30
    14. T. Kozai (1991) Photoautotrophic micropropagation., In Vitro Cell. Dev. Biol., Vol.27 ; pp.47-51
    15. T. Kozai , K. Iwabuchi , K. Watanabe , I. Watanabe (1991) Photoautotrophic and photomixotrophic growth of strawberry plantlets in vitro and changes in nutrient composition of the medium., Plant Cell Tissue Organ Cult., Vol.25 ; pp.107-115
    16. T. Kozai , Y. Koyama , I. Watanabe (1988) Multiplication of potato plantlets in vitro with sugar free medium under highphotosynthetic photon flux., Acta Hortic., ; pp.121-127a
    17. T. Kozai , C. Kubota , I. Watanabe (1988) Effects of basal medium composition on the growth of carnation plantlets in auto- and mixo-trophic tissue culture., Acta Hortic., ; pp.159-166b
    18. T. Kozai , H. Oki , K. Fujiwara (1990) Photosynthetic characteristics of Cymbidium plantlet in vitro., Plant Cell Tissue Organ Cult., Vol.22 ; pp.205-211a
    19. T. Kozai , K. Sekimoto (1988) Effects of the number of air changes per hour of the closed vessel and the photosynthetic photon flux on the carbon dioxide concentration inside the vessel and the growth of strawberry plantlets in vitro., Envrion. Control Biol., Vol.26 ; pp.21-29
    20. T. Kozai , A. Takazawa , I. Watanabe , J. Sugi (1990) Growth of tobacco seedlings and plantlets in vitro as affected by in vitro environment., Envrion. Control Biol., Vol.28 ; pp.31-39b
    21. H. Lee , S. Kim , M. Chung , C. Kim , J. Chung (2006) Effect of several culture conditions on growth of carnation propagules., Korean J Hort Sic Technol, Vol.24 ; pp.392-397
    22. Ministry of Agriculture, Food and Rural Affairs Database (2015) Key statistics of agriculture, food and rural affairs in 2015., http://www.mafra.go.kr/mafra/366/subview.do?enc=Zm5jdDF8QEB8JTJGYmJzJTJGbWFmcmElMkY3MSUyRjMwNDQ2OSUyRmFydGNsVmlldy5kbyUzRg%3D%3D
    23. M. Mousseau (1986) CO2 enrichment in vitro. Effect on autotrophic and heterotrophic cultures of Nicotiana tabacum (var. Samsun)., Photosynth. Res., Vol.8 ; pp.187-191
    24. T. Murashige , F. Skoog (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures., Physiol. Plant., Vol.15 ; pp.473-479
    25. SJ Murch , KL Choffe , JMR Victor , TY Slimmon (2000) Thidiazuron-induced plant regeneration from hypocotyl cultures of St. John’s wort plants (Hypericum perforatum cv ‘Anthos’)., Plant Cell Rep., Vol.19 ; pp.576-581
    26. Q.T. Nguyen , T. Kozai (2001) Photoautotrophic micropropagation of tropical and subtropical woody plants., Prog Biotechnol, Vol.18 ; pp.335-334
    27. M.M. Oh , H. Lee , J.E. Son (2009) Improvement of growth of potato (Solanum tuberosum L. cv. Dejima) plants at in vitro and ex vitro and energy efficiency by environmental control with growth stage in photoautotrophic micropropagation system., J Bio-Environ Con, Vol.18 ; pp.23-28
    28. F.R. Pretto , E.R. Santarem (2000) Callus formation and plant regeneration from Hypericum perforatum leaves., Plant Cell Tissue Organ Cult., Vol.62 ; pp.107-113
    29. I. Tichá , F. Čáp , D. Pacovská , P. Hofman , D. Haisel , V. Čapková , C. Schäfer (1998) Culture on sugar medium enhances photosynthetic capacity and high light resistance of plantlets grown in vitro., Physiol. Plant., Vol.102 ; pp.155-162
    30. Y. Xiao , T. Kozai (2004) Commercial application of a photoautotrophic micropropagation system using large vessels with forced ventilation: Plant growth and production cost., HortScience, Vol.39 ; pp.1387-1391
    31. Y. Xiao , G. Niu , T. Kozai (2011) Development and application of photoautotrophic micropropagation plant system., Plant Cell Tissue Organ Cult., Vol.105 ; pp.149-158
    32. Y Xiao , Y Zhang , K Dang , D Wang (2007) Growth and photosynthesis of Dendrobium candidum Wall. ex Lindl. plantlets cultured photoautotrophically. Prop Ornam Plants7:89-96,
    33. M. Zhang , D. Zhao , Z. Ma , X. Li , Y. Xiao (2009) Growth and photosynthetic capability of Momordica grosvenori plantlets grown photoautotrophically in response to light intensity., HortScience, Vol.44 ; pp.757-763
    34. S.M.A. Zobayed , F. Afreen-Zobayed , C. Kubota , T. Kozai (1999) Stomatal characteristics and leaf anatomy of potato plantlets cultured in vitro under photoautotrophic and photomixotrophic conditions., In Vitro Cell. Dev. Biol. Plant, Vol.35 ; pp.183-188
    35. S.M.A. Zobayed , P.K. Saxena (2004) Production of St. John’s wort plants under controlled environment for maximizing biomass and secondary metabolites., In Vitro Cell. Dev. Biol. Plant, Vol.40 ; pp.108-114